The present invention relates to nucleic acids encoding dog gastric lipase (DGL), and other polypeptide derivatives of the latter possessing a lipase activity, as well as their use, especially for the production of these polypeptides. The subject of the invention is also the polypeptides encoded by these nucleic acids, and the use of these polypeptides in pharmaceutical compositions.
DGL is a glycoprotein of about 380 amino acids (AA) of a molecular weight of about 49 kilodaltons (KD) synthesized in the form of a precursor containing a signal peptide at the amino-terminal (NH.sub.2 -terminal) end and secreted by the median cells of dog stomach fundic mucosa (Carriere F. et al. Eur. J. Biochem. 202 (1991) 75-83).
This enzyme belongs to a family of so-called "preduodenal" lipases of which certain members have already been purified and sometimes even cloned (Docherty A. J. P. et al., Nucl. Ac. res. 13 (1985) 1891-1903; Bodmer M. W. et al., Biochem. Biophys. Act. 909 (1987) 237-244; Moreau H. et al., Biochem. Biophys. Act. 960 (1988) 286-293; European Patents No. 0,191,061 and No. 0,261,016).
For a long time, it was taken for granted that the hydrolysis of food lipids occurred in the small intestine by virtue of the action of enzymes produced by the pancreas (Bernard C., C.R. Acad. Sci. 28 (1849) 249-253).
Observations suggested, however, that the hydrolysis of triglycerides could occur in the stomach by means of preduodenal enzymes (Volhard, F., Z. Klin. Med. 42 (1901) 414-429; Shonheyder, F. and Volquartz, K. Acta Physiol. Scand. 9 (1945) 57-67). These enzymes, and in particular dog gastric lipase, have enzymatic and physico-chemical properties which distinguish them from mammalian pancreatic lipases. These differences between gastric and pancreatic lipases essentially relate to the following points: molecular weight, amino acid composition, resistance to pepsin, substrate specificity, optimum pH for action, and stability in acidic medium.
Furthermore, in vitro, under certain conditions, a synergy of action between gastric and pancreatic lipases can be detected on the hydrolysis of long-chain triglycerides (Gargouri, Y. et Al., Biochem. Biophys. Act. 1006 (1989) 255-271).
Several pathological conditions (cystic fibrosis, pancreatic exocrine insufficiency) are known where the patients totally or partially lack pancreatic exocrine secretion and therefore the enzymes necessary for the hydrolysis of foods (amylases, lipases, proteases). The non-absorption of fats at the intestinal level, and especially long-chain triglycerides, results in a very substantial increase in steatorrhea in these patients and in a very substantial slowing down of weight gain in young patients. In order to overcome this, pig pancreatic extracts are administered to these subjects at the time of meals. The therapeutic efficacy of these extracts could be greatly improved by the co-prescription of DGL by virtue of its specificity of action on long-chain triglycerides.
The purification and the determination of the NH2-terminal sequence of DGL are described in the article by F. Carriere which appeared in Eur. J. Biochem. 201, 75-83, 1991. A process permitting the extraction of this enzyme from dog stomachs is also described in this publication. This process consists essentially in subjecting dog stomachs to an extraction by an acidic aqueous medium (pH 2.5); the-lipase extract is precipitated by addition of water-soluble salts, then by a filtration on a molecular sieve, followed by a separation by ion-exchange chromatographies, as well as by gel filtration, and an elution fraction containing the lipase is recovered. The purified DGL obtained by these processes has a molecular weight according to the Laemmli technique of 49,000 daltons, of which 6000 correspond to sugars and 43,000 to a protein.
Obvious reasons of difficulties of supply of dog stomachs prevent any development of this process both at the laboratory level and at the industrial level, hence the necessity to find a process avoiding the use of dog stomachs, which makes it possible to produce DGL in a large quantity.